Circulating placenta-derived extracellular RNAs (exRNAs) offer tremendous potential as safe, highly-informative, non-invasive biomarkers for placental function because they provide dynamic data regarding the functional, metabolic, inflammatory and perfusion state of the placenta and reflect the status of the placenta at a cellular level beginning in the first trimester. Thus, they can reflect problems before structural or end-organ effects have occurred and thereby enable risk-stratification, early intervention, and provide valuable insights into disease pathogenesis. However, widespread clinical application of exRNAs as biomarkers for placenta function has been limited by numerous technical limitations: (1) Existing exRNA isolation kits/protocols have poor recovery rates, size biases, do not inactivate the ubiquitous nucleases sufficiently to preserve the information- rich long coding transcripts, and are too costly and not amenable to automated application that would be required in a clinical setting, (2) placenta-derived exRNAs represent only a fraction of total exRNAs and hence sequencing depth has to be very deep in order for them to be adequately sampled, (3) the full repertoire of placental cell types and characteristic transcripts were not known and thus could not be fully queried and, (4) existing sequencing technologies have library preparation and sequencing times that are too long to permit turn-around- times with sufficient speed to provide results in a timely and actionable manner. In this grant, we assemble a multidisciplinary and multi-institutional team to develop four innovative new technologies that we pioneered that, when combined and optimized, offer an opportunity to solve these limitations and make exRNA a transformative clinical and research tool. In Aim 1, we use Automated exRNA isolation (AxRI), a novel method for automated, high-throughput, low cost, isolation of exRNA with near-instantaneous inactivation of nucleases and consequent protection of the information-rich coding RNA transcripts to isolate exRNAs from maternal circulation. We then use a customized capture assay, adapted from one we developed for cancer panel screening, to enrich placenta-derived transcripts using transcript information from our single-cell atlas of the human placenta. In Aim 2, we further develop our nanopore-based library preparation and sequencing technology to sequence the transcripts on a hand-held, disposable nanopore-based DNA sequencer at speeds that are much faster than existing next-generation technologies and that can deliver same-day results. In Aim 3, we apply the technologies developed in Aims 1 and 2 to generate and validate reference profiles for exRNA transcripts across gestation beginning in the early first trimester. Collectively, the development, optimization, validation, and combination of the technologies proposed in this grant will result in a clinically-viable and affordable platform that will provide an unprecedented ability to safely and non-invasively assess placenta function and composition at a near-cellular level and transform how we monitor and study pregnancy.